Microbes which invade the human body are challenged by several defense mechanisms. The nature of the defense mechanisms which any given microbe faces depends on the genetic makeup and the physiologic state of the host as well as the portal of entry of the invading microorganism.
Host defenses include mechanical factors and chemical factors. Mechanical factors which help protect epithelial surfaces include the washing action of bodily fluids, including tears, saliva and urine, trapping on mucous layers, removal by cilia and elimination by coughing, sneezing or desquamation. A further mechanical defense is offered by the physical integrity of the skin, although mucous membranes can be penetrated by some pathogens.
Chemical defense factors include the acidity of gastric secretions, unsaturated fatty acids on the skin which kill certain bacterial species, lysozyme in tears, saliva and nasal secretions, iron-binding proteins at the mucosal surface, transferrin in serum, and spermine in semen. Secretions of the mucous membranes also contain antibodies, especially those of the IgA class. Microbial antagonism between different potentially pathogenic bacteria, fungi and yeast strains occurs at the level of competition for nutrients and through the production of inhibitory substances; this antagonism affords further protection to the host.
If the barriers of the skin or mucous membranes are crossed, immunological factors (e.g., antibodies) which are specific to the microorganism as well as nonspecific cellular defenses come into play. In addition, there are some chemical factors which also play a role in host defense, especially transferrin, which chelates available iron on which microorganisms are dependent.
Nonspecific cellular defenses in the form of phagocytic white blood cells from local tissues and the bloodstream respond to an invading microbe. Polymorphonuclear leukocytes (PMNs) actively phagocytize particulates such as bacterial or fungal cells. PMNs are the first class of phagocytic cells recruited to the site of infection or inflammation. The PMNs contain azurophilic or primary granules, which contain lysosomal proteases, myeloperoxidase, lysozyme and certain antimicrobial proteins. Secondary granules within these cells contain alkaline phosphatase, lactoferrin and lysozyme. Stores of glycogen within the PMNs provides for energy through glycolysis so that the cell can function in an anaerobic environment.
Adherence of a particle to the surface of a phagocytic cell initiates phagocytosis; the particle enters the cytoplasm in a phagocytic vacuole. This triggers a respiratory burst and the generation of microbicidal metabolites; the primary granule fuses with the phagocytic vacuole to form a digestive vacuole called the phagolysosome. Intracellular killing of the ingested microorganism occurs as a result of oxygen-dependent and oxygen-independent mechanisms. The oxygen-dependent bactericidal halogenating system uses granule myeloperoxidase, hydrogen peroxide and chloride ion to kill bacteria and viruses via either halogenation of cellular or viral constituents or via reactive oxygen intermediates. Oxygen-dependent killing can also proceed by direct reduction of molecular oxygen via the cytochrome b-oxidase system (reviewed by Orkin (1989) Ann. Rev. Immunol. 7:277-308). Oxygen-dependent killing mechanisms are reviewed by Beaman and Beaman (1984) Ann. Rev. Microbiol. 38:27-48.
The primary granules contain three major groups of antibacterial proteins. The first group includes catalytically active proteins which are only weakly antibacterial when tested individually in purified form. Examples from this group include lysozyme, elastase and collagenase. These enzymes probably participate in the digestion of microorganisms killed by other mechanisms, but elastase, for example, is believed to potentiate killing by the halogenating system. The second category of granule proteins includes those with catalytic activity and bactericidal activity which is independent of the catalytic activity. An example is the chymotrypsin-like neutral protease of human neutrophils. A third group contains bactericidal members which lack known catalytic activity; such a protein class has been purified from rabbit neutrophils. Included in this class are defensins and cationic antibacterial proteins.
Some cationic antibacterial proteins are of relatively high molecular weight (greater than about 25 kDa) and kill certain Gram negative bacteria such as Escherichia coli, Salmonella typhimurium and Pseudomonas aeruginosa by damaging the cytoplasmic membrane, leading to increased membrane permeability. Human bactericidal/permeability increasing protein (BPI) is a strongly basic protein with molecular weight of about 59 kDa. It is believed that when bound to the outer membrane of susceptible bacterial cells, hydrophobic channels through the outer envelope are exposed, and as a secondary effect, there is a selective activation of autolytic enzymes including phospholipase and peptidoglycan hydrolases. Gram positive bacteria, certain Gram negative bacteria and fungi are not affected by BPI in vitro.
Low molecular weight cationic proteins (10 kDa to 25 kDa) have been reported which inhibit the multiplication of such Gram positive bacteria as Staphylococcus aureus (Root and Cohen (1981) Rev. Infect. Dis. 3:565-598). In addition, cationic proteins with fungicidal activity have been identified in alveolar macrophages. It is believed that cationic proteins are most efficient in killing phagocytized microorganisms in combination with other microbicidal defense mechanisms (Elsbach and Weiss (1983) supra).
Generally defensins are relatively small polypeptides of about 3-4 kDa, rich in cysteine and arginine. Gabay et al. (1989) Proc. Natl. Acad. Sci. USA 86:5610-5614, used reverse phase HPLC to purify 12 major polypeptides from the azurophil granules of human PMNs; purified proteins were analyzed individually for antimicrobial activity and for N-terminal amino acid sequence. A 4 kDa defensin (HNP-4) and a 29 kDa polypeptide named azurocidin were purified and shown to possess broad spectrum antimicrobial activity. Defensins as a class have activity against some bacteria, fungi and viruses. They are also reported to have cytotoxic activity against transformed cells. Selsted et al. (1985) J. Clin. Invest. 76:1436-1439, presents a sequence comparison of human and rabbit defensins. The defensins are believed to have molecular conformations stabilized by cystine infrastructure, which are essential for biological activity.
Granzymes are a family of serine proteases in the granules of cytolytic lymphocytes. Proteolytic enzymes are believed to function in cell-mediated cytoxicity; some of the genes have been cloned, and sequence information is available. Within the granzyme family there is at least 38% amino acid sequence identity. Human lymphocyte protease has 73% amino acid sequence identity to mouse granzyme B (Jenne and Tschopp (1988) Immunol. Reviews 103:53-71).
Cathepsin G (Cat G) is a granule protein with chymotrypsin-like activity; it is also known as chymotrypsin-like cationic protein. Cat G (Odeberg and Olsson (1975) J. Clin. Invest. 56:1118-1124) and three other mutually homologous polypeptides called defensins are active against a broad spectrum of gram positive bacteria, gram negative bacteria and fungi (Shafer et al. (1986) Infect. Immun. 54:184-188; Shafer et al. (1988) Infect. Immun. 56:51-53; Drazin and Lehrer (1977) Infect. Immun. 17:382-388; Ganz et al. (1986) Semin. Respir. Infect. 1:107-117). Sensitive bacteria include Capnocytophaga sputigena, Escherichia coli, Listeria monocytogenes, Neisseria gonorrhoeae, Pseudomonas aeruginosa and S. aureus. All of these pathogens, with the notable exceptions of P. aeruginosa and C. sputigena, are only sensitive to both enzymatically-active and -inactive cathepsin G (Miyasaki and Bodeau (1991) J. Clin. Invest 87:1585-1593; Wasiluk et al. (1991) Infect. Immun. 59:4193-4200 and Table 11 herein). P. aeruginosa and C. sputigena are only sensitive to enzymatically-active cathepsin G. It is not clear, however, if cathepsin G-killing of these two pathogens requires degradation of bacterial proteins or whether an intact active site is needed to align antibacterial domains of cathepsin G with the bacterial target.
Gabay et al. (1989) supra, has reported antibacterial activities of a number of proteins isolated from human PMNs, including cathepsin G and elastase, and has given the amino terminal sequence of these and other proteins. The N-terminal five amino acids of elastase and Cat G are identical; further sequences have significant relatedness. Cat G also exhibits significant sequence similarity to chymotrypsin, which is not known to exhibit antimicrobial activity similar to that of Cat G.
The sequence of human Cat G is known, and the gene has been cloned from human leukemic cell line U937 (Salvesen et al. (1987) Biochemistry 26:2289-2293). Sequence analysis of the cDNA revealed significant sequence identity to rat mast cell proteinase (47%) and to an activated mouse cytotoxic lymphocyte product (56%).
Another class of antimicrobial polypeptides are those known as magainins; at least five proteins can be isolated from the skin of the African clawed frog (Xenopus laevis). The natural proteins are active against a broad range of microorganisms including bacteria, fungi and protozoans (Zasloff (1987) Proc. Natl. Acad. Sci. USA 84:5449-5453). The broad spectrum antimicrobial activity is present in synthetic peptides and in certain truncated analogs of the natural proteins. Derivatives of about 19 to about 23 amino acids have antibacterial activity as measured using Escherichia coli. In the protozoan Paramecium caudatum treated with the magainin peptides, there is disruption of membrane functions. The configurations of the bioactive peptides can be modeled as amphiphilic alpha-helices and are sufficiently long to span a lipid bilayer. (Zasloff et al. (1988) Proc. Natl. Acad. Sci. USA 85:910-913). Spanning a lipid bilayer is believed to require at least 20 amino acid residues in an alpha-helical configuration (Kaiser and Kennedy (1987) Ann. Rev. Biophys. Chem. 16:562-581). The sequence of a representative magainin peptide is GIGKFLHSAKKFKAFVGEIMN (SEQ ID NO:48) (Zasloff et al. (1988) supra).